Purpose: :
Information transfer in mammalian horizontal cells(HC) is poorly understood. The occurrence of clear–corevesicles, the vesicular GABA transporter, and several synapticand vesicular proteins in HCs suggest transmitter release ismediated by a vesicular mechanism. In the mammalian presynapticnerve terminal, formation of a complex between VAMP1 or VAMP2,syntaxin1 and SNAP–25 is known to mediate fusion of thevesicle and target membranes. To determine whether the molecularmachinery for vesicular transmitter release is present in HCs,we examined the distribution of one component of this complex,the vesicle–associated membrane proteins (VAMP), in theouter retina.

Methods: :
Immunohistochemistry with antibodies (AB) to all 7known isoforms of VAMPs, and to the VAMP–like proteinstomosyn and amisyn were used to evaluate mouse outer retina.Double labelings were performed with ABs to calbindin D–28k,a HC marker, and to vesicular glutamate transporter 1 (VGLUT1)or PSD95 to identify photoreceptor (PR) terminals. Immunostainingwas evaluated using confocal microscopy.

Results: :
Strong VAMP1 immunoreactive puncta embedded in thesynaptic triad of the PR terminals were observed in the outerplexiform layer (OPL) using a VAMP1–specific AB (ABCAM).Double label studies showed that these puncta were in the tipsof HC dendrites and axons, although we could not eliminate thepossible expression of VAMP1 in PR synaptic ribbons due to theirproximity. In contrast, strong immunoreactivity was in PR terminalsusing a second VAMP1 AB (Synaptic Systems), presumably recognizingVAMP1 and VAMP2, and two different VAMP2 ABs. The presence ofVAMP1 or VAMP2 immunoreactivity in HC tips could not be eliminatedbecause they are deeply embedded in PR terminals and immunoreactivitywas distributed across the entire terminal. Weak VAMP4 immunoreactivity(ABCAM), compared to VAMP1 and VAMP2, was also in PR terminals.Moreover, PR terminal immunostaining was absent using a secondVAMP4 AB (Synaptic Systems). VAMP3 immunoreactivity was in bipolarcell dendrites, and weak tomosyn immunoreactivity was in theOPL, although it was not possible to distinguish if tomosynwas colocalized with HC tips or PR terminals. No immunostainingwas observed in the OPL using VAMP5, VAMP7, VAMP8 and amisynABs.